Summary The ~200 mammalian enzymes belonging to the cullin-RING ubiquitin ligase (CRL) family permeate virtually every aspect of biology. Their combinatorial architecture where multiple substrate receptors (SRs) compete for access to cullin cores creates the fundamental problem of assembling CRL complexes through high affinity protein interactions while maintaining the flexibility to dynamically sample the entire SR repertoire. Studies over the past decade ? many pioneered by the applicant laboratories - have revealed that this problem is addressed by a CRL assembly cycle directed by the reversible modification of cullins with the ubiquitin-related peptide NEDD8. The model that has emerged postulates that removal of NEDD8 by the COP9 Signalosome (CSN) renders CRLs sensitive to CAND1, a factor that promotes the assembly of newly synthesized SRs with CRL core complexes. Substrate, in turn, will trigger cullin neddylation to stabilize and activate a cognate CRL complex. The cycle resets with CSN-mediated NEDD8 removal. Notwithstanding its explanatory power, essential pieces of this model remain either unproven or entirely obscure: What affords CSN's ability to bind to ~200 structurally diverse CRL? How is this molecular recognition directed by post-translational modifications? What is the mechanism for release of CRLs from the CSN? Recently published work from the applicant laboratories has provided several unique entry points into these important questions that have coalesced to a uniform project: Dr. Sharon's lab has identified a new subunit of the CSN, CSNAP, and showed that it flexibly tethers distinct subassemblies thus potentially affording the plasticity required for promiscuous CRL binding. The demonstration by Dr. Wolf's group that CAND1 promotes the exchange of SRs from cullins raised the intriguing possibility that CAND1 integrates the release of SRs from CRLs with the release of CRLs from the CSN in a single step. However, despite their obvious importance in CRL control, the biochemical functions of CSNAP and CAND1, both in vitro and in vivo, remain enigmatic. Synergizing through their unique but complementary technological expertise in quantitative mass spectrometry, the investigators will jointly address novel mechanisms of CSNAP and CAND1-mediated CRL control.